Biofilm

AimsPseudomonas aeruginosa biofilm-associated infections pose a significant clinical challenge due to their inherent antibiotic tolerance. This study aimed to evaluate the antibacterial and antibiofilm activity of Placentrex, a standardised aqueous placental extract, against P. aeruginosa and to elucidate its molecular mechanism of action using RNA sequencing (RNA-seq). Methods and ResultsPlacentrex exhibited potent bactericidal activity against P. aeruginosa at 50 mg/mL. Biofilm formation was significantly inhibited by [~]87% at 50mg/mL after 72 hours. Preformed biofilms were eradicated by [~]93% and [~]89% at 50 and 25 mg/mL, respectively. Interestingly, biofilm viability was reduced by [~]93% and [~]87% upon treatment with 50 mg/mL and 25 mg/mL of Placentrex, respectively. EPS characterisation revealed that the EPS contain a single large polysaccharide, and chromatography data suggested that it is made up of glucose as a monomer. RNA-seq identified coordinated downregulation of seven key genes, namely, flp major pilin (surface attachment), extracellular solute binding protein (ABC transporter-mediated nutrient sensing and biofilm maintenance), gntP permease (carbon metabolism), AraC family transcriptional regulator (quorum sensing and polysaccharide biosynthesis), ureE (urease nickel metallochaperone), aromatic amino acid permease (pyoverdine and PQS biosynthesis), and MFS transporter (efflux and autoinducer export). ConclusionsPlacentrex exerts comprehensive antibiofilm and antibacterial activity through simultaneous disruption of surface attachment, nutrient-sensing-driven biofilm maintenance, quorum sensing, carbon metabolism, urease virulence maturation, and efflux-mediated persistence. This polypharmacological mechanism supports Placentrex as a promising multi-target antibacterial agent against P. aeruginosa biofilm-associated infections. Impact statementPlacentrex is a potential anti-biofilm agent against Pseudomonas aeruginosa.

Biofilms pose a significant challenge to antimicrobial therapy. Bacteria in biofilms differ from planktonic counterpart in their altered metabolism, collective behavior, protective role of extracellular matrix and diversified microbial subpopulations. These attributions significantly influence bioavailability and activity of antibiotics. The presence of bacterial aggregates during acute infections expands the problem to many other conditions previously not discussed in the biofilm context. Klebsiella pneumoniae is a leading cause of life-threatening hospital-acquired infections and is included in the WHO Bacterial Priority Pathogens List due to increasing antimicrobial resistance. The combination of antimicrobial resistance and the ability to form biofilms severely limits the efficacy of antibiotic treatments. In this study, we investigated the in vitro susceptibility of mature biofilms to 13 antimicrobials of K. pneumoniae clinical isolates from a single hospital. The resistance profiles of the local clinical isolates were consistent with the global epidemiology of K. pneumoniae. Minimal biofilm eradication concentrations (MBEC) for mature biofilms were defined with two assays (biomass and metabolic activity measurements) and brought into relation with susceptibility breakpoints and plasma (Cmax). Colistin sulfate, tigecycline, cephalosporins and combination of imipenem with cilastatin were the most potent biomass eradicators, while suppression of metabolic activity was barely reachable. Moreover, we observed a notable increase in metabolic activity upon exposure to sub-MBEC concentrations of antibiotics. Finally, our data broach a subject of antibiotic prioritization with respect to biofilm tolerance. IMPORTANCEThis study addresses the critical gap between standard antibiotic susceptibility testing and the tolerance of biofilm and microbial aggregates during infections caused by K. pneumoniae. By systematically evaluating mature biofilms from a significant number of clinical isolates, we demonstrate that colistin and tigecycline show potent activity against both biofilm biomass and metabolic activity, whereas cephalosporins primarily reduce biomass without effectively suppressing bacterial metabolism, and other drugs have only weak effects on biofilms at clinically achievable concentrations. Furthermore, the alarming observation that sub-inhibitory biofilm eradication concentration (sub-MBEC) of antibiotic can paradoxically increase the metabolic activity of biofilms highlights a potential risk factor for therapy failure and resistance development. Our findings contribute to the necessary evidence base for prioritizing existing antibiotics in the limited armamentarium against biofilm-forming K. pneumoniae.

Enterococcus faecalis is a Gram-positive intestinal commensal and opportunistic pathogen capable of causing serious infections, including urinary tract infections, endocarditis, and wound infections. A major contributor to its persistence during infection is the ability to form biofilms on host tissues and medical devices. Biofilm cells have higher phenotypic tolerance to antimicrobial treatment than planktonic bacteria. While mechanisms governing biofilm assembly in E. faecalis have been widely studied, the processes that regulate biofilm dispersion, the final stage of the biofilm life cycle, remain poorly understood. In this study, we found that dispersion is triggered by a tenfold step-change increase in nutrient availability and by cell free supernatant (CFS) of E. faecalis OG1RF cultures. Cells released from biofilms regain sensitivity to antibiotics similar to planktonic cells but maintain a high potential for adherence. We characterized the glycosyltransferase epaOX, which contributes to the structure of the enterococcal polysaccharide antigen as necessary for nutrient step-change induced dispersion, CFS induced dispersion, and adhesion of dispersed cells. Supplementation of epaOX mutant CFS with galactose and N-acetylgalactosamine was sufficient to restore CFS induced dispersion. Together these data suggest that dispersion in OG1RF occurs with fast kinetics, affects antibiotic sensitivity and is regulated in part by known virulence factors. ImportanceE. faecalis causes difficult to treat infections at numerous body sites in human patients. E. faecalis biofilms are adherent populations that require high levels of antibiotics for treatment. Biofilms undergo a disassembly process named dispersion that allows individual cells to leave the biofilm and colonize new locations. Dispersed cells in other species are killed by lower amounts of antibiotics than biofilm cells. Here we showed that dispersion occurs in E. faecalis and lowers the level of antibiotics needed to kill dispersed cells. Dispersion triggers could be used in the future to design treatments that increase the effectiveness of antibiotics.

The evolution of antimicrobial resistance (AMR) in chronic biofilms is often viewed as a unidirectional path toward higher fitness, yet the metabolic constraints governing these trajectories remain poorly understood. We performed a four-passage evolution experiment using a murine lung biofilm model to assess the impact of prolonged ciprofloxacin (CIP) exposure on resistance and host response. This approach integrated population-level adaptive dynamics, whole-genome sequencing (WGS), and NMR-based metabolomics, alongside histopathology and cytokine analysis. Prolonged CIP treatment accelerated resistance, with isolates reaching MICs of 8-12 mg/L (a 32- to 48-fold increase) by the fourth passage. WGS revealed distinct evolutionary trajectories: control isolates accumulated metabolic and regulatory mutations without susceptibility changes, while CIP-treated isolates exhibited a stepwise progression from metabolic adaptation to high-level resistance, marked by early nfxB and late gyrA mutations. Metabolomic profiling revealed progressive divergence, with PCA identifying the nfxB genotype as the primary driver of variation (49.1% of variance). This resistant metabolic state was characterized by the depletion of central carbon metabolites, including glucose and tyrosine, alongside the accumulation of essential amino acids. Importantly, these changes were accompanied by a distinct trade-off; high-level CIP resistance triggered collateral sensitivity to tobramycin and aztreonam. While CIP treatment ultimately reduced neutrophilic inflammation (p = 0.011) and mucin production (p = 0.0496), early-passage lungs exhibited transient elevations in pro-inflammatory cytokines (CXCL2, MMP2, TNF-). In conclusion, the adaptive trajectory to CIP resistance involves metabolic rewiring and collateral sensitivity, offering a framework to exploit the evolutionary costs of resistance in chronic biofilm infections.

Pseudomonas aeruginosa is an opportunistic human pathogen that is hospital-endemic, forming biofilms on medical equipment and causing thousands of hospital-acquired infections each year. The success of P. aeruginosa as an opportunistic pathogen is linked to its phenotypic and genotypic adaptability. Relatedly, P. aeruginosa forms a small colony subpopulation in response to stresses like oxygen limitation and antibiotic exposure. Additionally, P. aeruginosa coordinates population-level decisions using a mechanism of cell-cell communication called quorum sensing. P. aeruginosa uses these signaling pathways to control virulence factor production and biofilm formation in the host. We show that certain quorum-sensing mutations promote phenotypic variation; specifically, deletion of lasR and autoinducer modulating mutations in rhlI enhanced small colony formation in a time course-dependent manner. Using transcriptome analyses of isogenic small and large colony variants, we show that small colony formation is driven in part by the PhoPQ two-component signal transduction system in quorum-sensing mutant backgrounds. Specifically, our data show that unphosphorylated PhoP represses rhlR gene expression, and that subsequent de-repression of quorum sensing contributes to the production of virulence factors and the small colony phenotype. In total, these findings provide insight on how mutations evolved by clinical strains might serve as a bet-hedging strategy to promote the formation of a small colony phenotype and alter quorum-sensing signaling within a subpopulation of a community.

Polydimethylsiloxane (PDMS) is an excellent material for the construction of biomimetic leaf replicas which reproduce leaf surfaces with high fidelity. This allows for the study of leaf surface-colonizing bacteria and the impact of the leaf topology on bacterial distributions and behavior. However, their application is limited to short-term experiments, as long term survival of microorganisms on their surface is not possible due to a lack of nutrient replenishment. On living leaves, nutrients diffuse across the cuticle via leaching, a process not yet replicated in biomimetic systems. Here, we explore whether water and fructose can be supplied to microbial colonizers on PDMS membranes by mimicking leaching. We created hybrid membranes by incorporating polymers (Carbopol, Pemulen, cellulose microfibers, cellulose nanocrystals, and polyvinylpyrrolidone) to enhance nutrient transport. We determined that bulk diffusion of water correlated negatively with membrane thickness and positively with polymer concentration. Further, fructose diffusion across hybrid membranes reached similar rates compared to isolated Populus x canescens leaf cuticles. Under high relative humidity, these membranes supported long-term bacterial survival. Our findings represent important steps towards the development of topomimetic leaf surfaces that sustain microbial life, enabling further investigation into the microbe-microbe interactions that take place on leaves.

BackgroundThe female lower reproductive tract harbors a complex microbiome that plays a critical role in reproductive health. A vaginal microbiome dominated by Lactobacillus crispatus (LC; Community State Type (CST) I) supports vaginal health, whereas a microbiome enriched with anaerobic species, such as Gardnerella vaginalis (GV) and Mobiluncus mulieris (MM) (CST IV) is linked to bacterial vaginosis (BV) and adverse outcomes, including sexually transmitted infections, infertility, and preterm birth. Although antibiotics such as metronidazole and clindamycin are commonly prescribed to treat BV, recurrence rates remain high, and the impact of these treatments on bacterial extracellular vesicles (bEVs), critical mediators of host-microbe interactions, is poorly understood. ResultWe investigated how antibiotic treatment at a dose below minimum inhibitory concentration alters the production and immunomodulatory function of bEVs derived from GV, MM, and LC. Using nanoparticle tracking analysis, cytokine profiling, and TLR pathway analyses, we found that antibiotic treatment significantly enhanced the inflammatory properties of bEVs in a species- and antibiotic-specific manner. Notably, bEVs from antibiotic-exposed GV and MM cultures induced elevated cytokine responses in epithelial and immune cells, primarily through TLR2 activation for GV bEVs, and through both TLR2 and TLR5 activation for MM bEVs. While LC bEVs are typically non-inflammatory, exposure to metronidazole, even at a lower dose than what is used clinically, rendered them immunostimulatory, suggesting a potential unintended proinflammatory consequence of treatment on beneficial microbes. We also detected bEVs in human vaginal swabs, including vaginolysin-positive bEVs, even in CST I microbiomes, indicating that low-abundance microbes, including pathogens, remain transcriptionally active. ConclusionsThese findings suggest that antibiotics not only reduce microbial load but also reshape bacterial communication via bEVs, potentially contributing to inflammation, epithelial barrier disruption, persistent dysbiosis, and recurrent infections. This work underscores the need for precision antimicrobial strategies that eliminate pathogens while preserving beneficial bacteria and their functional bEVs. Future therapies may benefit from considering the ecosystem-wide effects of antibiotics on the vaginal microbiome and its bEV-mediated signaling network.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated real-time kinetic analysis framework integrating connectivity-based segmentation, automated baseline alignment, and robust sliding-window algorithms to quantify the biomineralization competence of Bacillus subtilis under varying calcium regimes. Crucially, our results demonstrate that calcium carbonate promotes microbial growth as effectively as the highly soluble calcium acetate, providing strong evidence that B. subtilis actively solubilizes this crystalline powder to facilitate its metabolic requirements. Despite this growth efficacy, we found that calcium carbonate is an inadequate source for macro-calcite production compared to organic salts. By quantifying the expression efficiency of the sinI reporter gene, we determined that calcium-acetate-driven ECM expression significantly enhances the structural compatibility required for robust biomineralization. Furthermore, kinetic modeling suggests that ECM overproduction can partially compensate for defects in crystal growth-when provided crystalline calcium carbonate powder. These findings, enabled by high-resolution automated signal processing, underscore the critical role of self-mediated carbonate supply and present new engineering pathways for upcycling mineral-rich construction waste.

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to chronic infection and mucus retention in the lungs, with lung function gradually deteriorating through recurrent pulmonary exacerbations (PEx). Virulence factors (VFs) of Pseudomonas aeruginosa and Staphylococcus aureus are thought to contribute to pulmonary exacerbations. Our study objective was to identify VF genes related to PEx, high Pseudomonas abundance, and high Staphylococcus abundance in persons with CF (pwCF). MethodsThis was an ancillary study of pwCF treated with IV antibiotics for PEx between 2016-2020 at Childrens National Hospital. Using shotgun metagenomics and ShortBRED, we identified bacterial VF genes and used DESeq2 to determine differential expression of VF genes across comparators. ResultsTwenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years, 41% female, 59% white, 36% Hispanic, and 45% had an F508del homozygous CFTR mutation. Minimal differences in VF gene abundance were identified across clinical state. The most differentially increased VF genes found in Pseudomonas high samples were associated with an aminotransferase (log2FC 25.9), flagellar biosynthesis (log2FC 8.3), and type VI secretion systems (log2FC 8.2). The most differentially increased VF genes found in Staphylococcus high samples were an exotoxin (log2FC 26.7), macrolide phosphotransferase (log2FC 25.8), pathogenicity island proteins (log2FC 25.2 and 24.7), and VOC family proteins (log2FC 24.8). ConclusionsThese findings demonstrate that specific VFs associated with immune modulation, motility secretion systems, bacterial motility, and antibiotic resistance are related to P. aeruginosa and S. aureus abundance, providing potential targets for more personalized antimicrobial interventions.

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven cyanine monomer and dimer dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms.

1.The secondary messenger cyclic di-GMP is a ubiquitous bacterial signal that regulates the switch from a free-swimming to a sessile biofilm-forming lifestyle. Many biofilm-forming Pseudomonas species possess numerous c-di-GMP-binding proteins (CDGs) which regulate gene expression, protein activity, and protein complexes. However, the mechanisms by which numerous CDG effectors form a coherent signaling network to coordinate lifestyle changes remain poorly understood. We addressed this knowledge gap by focusing on ten CDG proteins involved in biofilm development in P. fluorescens SBW25. We used an integrated approach combining a protein interaction network from genome-wide yeast two-hybrid (Y2H) screens with large-scale biofilm and motility phenotype analyses via CRISPR interference (CRISPRi). Our network associated c-di-GMP signaling with processes such as signal transduction, solute transport, secretion, virulence, transcriptional regulation, DNA repair, and cell division. We discovered unknown functions of two CDG proteins in DNA repair and cell division, supporting the significance of our network. Notably, the phosphodiesterase DipA interacts with numerous CDG proteins through GGDEF domains. Phenotypic analyses revealed that CDG partners were highly correlated or strongly anticorrelated with DipA. These findings suggest that DipA is a central hub for CDG interactions that integrates opposing modules. These findings support the hub-based model of c-di-GMP signaling, which is crucial for localized control and rapid adaptation to environmental changes.

BackgroundWhether the lung microbiome represents a stable microbial colonization or a transient ecosystem shaped by continuous microbial turnover and controlled by host immunity remains unresolved. The murine lung microbiome largely consists of species from the former Lactobacillus genus with Ligilactobacillus murinus as a dominant species, bacterial genera such as Streptococcus, Staphylococcus, Mammaliicoccus, Enterococcus and other less frequently detected bacteria. Here, we directly addressed the question of persistence and host interaction of a dominant murine lung commensal in vivo and focused on the host immune response towards lung commensal bacteria. ResultsWe developed a transformation strategy for stable genomic integration of a green fluorescent protein (GFP)-encoding gene to track the fate of a lung bacterium. Following intranasal administration of GFP-labeled L. murinus in mice, bacteria were readily detected in the lungs at early time points but declined rapidly and became undetectable after 72 hours, as determined by quantification of viable bacteria and qPCR. Flow cytometry and fluorescence imaging revealed efficient uptake of GFP-labeled bacteria by lung phagocytes. These findings indicate that even dominant members of the murine pulmonary microbiota normally detected at low abundances are transiently present in the lungs without causing infection. We further analyzed the effects of moderate and high bacterial concentrations. While moderate bacterial loads were efficiently controlled without clinical effects, high concentrations induced severe lethargy, indicating a threshold-dependent host response. Finally, we demonstrated that pulmonary commensals such as L. murinus, Staphylococcus xylosus, and Mammaliicoccus sciuri, as well as conidia of the opportunistic lung pathogen Aspergillus fumigatus, are phagocytosed at comparable rates in macrophage assays. ConclusionsOur data demonstrate that even lung-adapted bacterial species fail to establish stable colonization and are instead subject to rapid immune-mediated elimination contributing to the maintenance of a low microbial burden in the lungs. While this homeostatic balance supports health, elevated bacterial loads trigger immune activation and, at high levels, lead to health deterioration. Together, these results support a model of a highly dynamic and transient lung microbiome, maintained by continual microbial immigration rather than long-term colonization. Accounting for the lung microbiome dynamics is essential for understanding host-microbiota interactions and respiratory health.

Patescibacteria are an elusive linage of "microbial dark matter" bacteria predicted to represent [~]25% of total bacterial diversity. Despite this abundance and ubiquity, these organisms are challenging to cultivate, resulting from their specialized episymbiotic lifestyle. All cultivated representatives to date, predominantly composed of Saccharibacteria from the oral microbiome, depend on cognate prokaryotic hosts for growth and reproduction. Studying the growth dynamics of episymbiotic bacteria and their hosts in batch cultures has suggested that many episymbionts initially reduce host populations, and that hosts eventually adapt to episymbiont stress after serial passaging. However, discontinuous batch cultures do not reflect natural interactions between these organisms due to their drastically different growth rates. An episymbiont requires several ([~]2-4) serial passages alongside its host to reach the high cell densities needed to impact host growth, which complicates investigation of host inhibition and adaptation to episymbiont stress. To describe these dynamics accurately, we utilized continuous culture via small-scale Raspberry Pi powered bioreactors, called Pioreactors. Within a bioreactor, host bacteria can be cultivated at a consistent growth rate indefinitely, providing the perfect substrate for cultivation of model Saccharibacteria. Quantification of time until host crash, crash severity, time until recovery, and stable co-culture density provides mechanistic ways to describe episymbiont-host interactions. First, we used these techniques to compare episymbiont infection by three different episymbionts, revealing distinct infection patterns ranging from mild inhibition with rapid host adaptation, to rapid host collapse followed by "arms race" oscillation dynamics. Then, bioreactors were used to quantify the episymbiotic role played by a known host-binding type 4 pili (T4P-2), demonstrating that loss of long-distance host binding significantly delayed the host crash without altering general crash dynamics. These experiments reveal that episymbionts can have drastically different effects on bacterial communities and provide the tools necessary to describe strain/species differences and molecular interactions. ImportanceEpisymbiotic Patescibacteria represent one of the largest branches of life on Earth, as well as one of the least understood. Furthermore, because Patescibacteria can manipulate their hosts growth and morphology they have immense ecological potential to be shaping the communities they occupy, both environmental and microbiome-associated. Our study highlights for the first time the potential of small-scale continuous cultivation for studying episymbiotic interactions that cannot be captured in discontinuous cultures. Herein we used these techniques to interrogate inter-species variation in host inhibition potential and to determine how loss of a long-distance episymbiosis factor mechanistically alters the cycle of episymbiont infection; however, this cultivation platform will enable researchers to answer many new questions about these ubiquitous host-episymbiont interactions.

Pathogen cross-contamination during food production is primarily controlled through environmental sanitation. However, sanitizer efficacy is often studied in bench-scale experiments that poorly approximate the fluid dynamics of sanitization and limits our understanding of commercial sanitization efficacy. This study paired computational fluid dynamics (CFD) estimates of shear stress with experimental measurements of Listeria innocua reduction on stainless steel following treatment with 100 ppm hypochlorite sanitizer. At the pilot-scale, sanitizer spray manually applied by researchers achieved a 2.6 {+/-} 0.4 log CFU/surface reduction; however, microbial reduction from manual operation of sanitizer spray equipment differed significantly between researchers (p < 0.05). Microbial reduction varied by location following stationary, bench-scale spray application of sanitizer for 3 s. The greatest reduction was at the point of sanitizer spray impingement (7.5 {+/-} 0.5 log CFU/surface) and directly adjacent to the impingement point (6.4 {+/-} 0.7 log CFU/surface) where shear stress was the highest. Significantly less microbial reduction (0.4 {+/-} 0.1 log CFU/surface) occurred where shear stress was lowest in the fluid-film of sanitizer running down from the impingement point (p < 0.05). Static submersion of inoculated coupons in sanitizer for 3 s resulted in a log reduction of 2.3 {+/-} 0.1 log CFU/surface. Discrepancies between bench-scale spraying, pilot-scale spraying, and submerged coupons demonstrate the need for sanitizer efficacy testing under realistic conditions to better estimate the risk reduction achieved through sanitation programs. IMPORTANCESanitation is critical for controlling pathogen cross-contamination during food production. These findings highlight the limitations of traditional approaches to sanitizer efficacy testing, not because they are invalid, but because they do not reflect the level of microbial reduction typically achieved in application. We demonstrate that these differences in outcomes are attributable to fluid dynamics and exposure, which are not well approximated in submerged coupon experiments. Accurate estimation of microbial reduction from sanitizer application is needed to guide food safety policy decisions. For example, overestimation of the risk reduction conferred by sanitizer treatment may result in food safety policies that neglect other sources of microbial reduction within sanitation programs.

Whether pathogen virulence influences antibiotic efficacy independently of bacterial growth dynamics has not been directly tested under controlled in vivo conditions. Here, we used the Galleria mellonella larval infection model to address this question. We first compared Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus across a wide range of inoculum sizes, revealing pronounced pathogen-specific differences in virulence. Despite these differences, all pathogens followed a similar infection trajectory characterised by an initial reduction in bacterial burden followed by successful proliferation within the host. Focusing on P. aeruginosa and E. coli, which differ markedly in virulence but display comparable in vivo growth rates (doubling times of approximately 30 minutes), we assessed antibiotic efficacy under experimentally controlled, growth-matched conditions. Treatment with ciprofloxacin or gentamicin at defined multiples of the MIC reduced bacterial loads, prevented host death, and cleared infections to an indistinguishable extent for both pathogens. These findings demonstrate that pathogen virulence does not determine antibiotic efficacy when bacterial growth is comparable in vivo, and support the predictive value of MIC-based susceptibility testing during active infection.

Resistance to a particular antibiotic can make bacteria sensitive to others, a phenomenon known as collateral sensitivity (CS). This study explored potential CS in clinical and experimentally evolved drug-resistant Pseudomonas aeruginosa (PA) and investigated underlying mechanisms. Whole-genome sequencing and RNA-seq were analyzed to identify genetic and transcriptional correlations. In vitro efficacies were assessed with co-and sequential-exposure regimens. Multiple CF isolates and experimentally evolved gentamycin (GEN) resistant strains consistently exhibited strong CS to novobiocin (NOV). Comparative genomics revealed pmrB gain-of-function mutations, which was further supported by transcriptomic signatures of pmrAB activation. Transcriptomic data suggests potential outer-membrane remodeling characterized by polyamine accumulation and compromised porin channel expression. Additionally, the reduction in proton motive force (PMF) further explains the possible mechanism underlying GEN resistance. As NOV efflux is PMF-dependent, this energetic deficit created a PMF-efflux mismatch, leading to hypersensitivity to NOV. Notably, sequential GEN[-&gt;]NOV treatment effectively restricted the emergence of GEN resistant subpopulations. Overall, our data suggest GEN resistance in PA may arises through envelope remodeling and reduced PMF, which impairs efflux pumps and creates hypersensitivity to NOV. Exploiting this PMF-efflux mismatch with sequential treatment effectively restricted the emergence of GEN resistance.

Airborne transmission of respiratory pathogens depends on their ability to remain viable in drying respiratory droplets, yet the physicochemical drivers of bacterial inactivation during droplet evaporation remain poorly quantified. This study combines controlled droplet experiments with physicochemical modeling to investigate how osmotic pressure dynamics influence bacterial survival. Using Escherichia coli and Staphylococcus epidermidis as Gram-negative and Gram-positive surrogates, respectively, we measured viability loss in artificial saliva droplets dried at multiple relative humidities and reconstructed the time-resolved osmotic pressure using the Respiratory Aerosol Model (ResAM). Both organisms remained stable while droplets were liquid but lost viability following efflorescence, when rapid solute concentration changes produced sharp osmotic pressure increases. The extent of inactivation scales log-linearly with the rate of osmotic pressure change around efflorescence: E. coli decays faster than S. epidermidis, and relationships derived in artificial saliva predict survival in independent phosphate-buffered saline experiments. A more rapid drop in humidity led to more severe osmotic shocks and greater inactivation. These results identify the rate of osmotic pressure change during efflorescence as a quantitative, medium-independent predictor of bacterial survival in drying respiratory droplets. ImportanceAirborne infection risk depends on how long microorganisms remain viable in respiratory particles after exhalation, yet the physical mechanisms controlling bacterial survival during droplet drying are not well defined. Evaporation of respiratory droplets concentrates salts and can impose sudden and extreme osmotic stress on microbes, but this process has been difficult to quantify because osmotic pressure cannot be measured directly inside microscopic droplets. Integration of droplet experiments with a physicochemical aerosol model shows that bacterial inactivation is governed primarily by the rate of osmotic pressure increase during droplet efflorescence rather than by static values of humidity or solute concentration alone. This mechanism explains why rapid drying may produce strong inactivation.

Sewer biofilms represent dynamic interfaces for exchange of bacteria and antibiotic resistance genes between biofilms and the overlying wastewater. Using inline, biofilm reactors, the movement of bacteria and 16S rRNA and carbapenemase genes (blaKPC, blaVIM, blaNDM, blaOXA-48-like, and blaIMP) between wastewater and sewer biofilms was investigated. Established, complex biofilms without these {beta}-lactamase (bla) genes, absorbed resistant bacteria within two minutes of exposure to high concentrations of resistant cultures in lab settings. Carbapenem-resistant organisms from these high-concentration source biofilms transferred to downstream biofilms over 60 minutes of representative sewer shear flows. Mass balances of bacteria and genes in biofilms versus wastewater under representative shear flow showed that biofilms exposed to resistant cultures contributed more to the wastewater than to the downstream biofilms. In field studies, established, complex biofilms without target carbapenem-resistant bacteria and genes from wastewater within hours and then stabilized between 2 to 15 days, not varying by more than 0.5 MPN/cm2 or 0.5 log gene copies (GC)/cm2. In contrast, metagenomic profiles of the bacterial community species continued to change up to 21 days. Established biofilms with resistant bacteria and genes exposed to tertiary-treated wastewater without target carbapenemase genes or meropenem antibiotics did not lose resistant genes or bacteria over nine days of exposure (i.e., < 1 log GC/cm2 reduction). Results show that sewer biofilms contribute to the resistance-gene signal found in sewer wastewater by absorbing and releasing bacteria and genes. Consideration of sewer biofilm dynamics is essential for more accurately interpreting wastewater bacterial concentrations in wastewater-based epidemiology studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/726639v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19f6ce0org.highwire.dtl.DTLVardef@1a507c8org.highwire.dtl.DTLVardef@1a2013dorg.highwire.dtl.DTLVardef@ff8613_HPS_FORMAT_FIGEXP M_FIG C_FIG

Corals harbor a diverse bacterial community that facilitates adaptation and sustains their health. In coral holobiont research, culture-independent approaches have transformed the existing paradigm. Molecular techniques, such as metabarcoding, revealed a high diversity of previously unrecognized bacterial symbionts. Coral microbiota characterization has relied on these techniques over the last decade, but relying solely on them does not provide a detailed understanding of the dynamics of the coral holobiont complex. Returning to classic microbiological methods and in vitro experimentation can yield novel insights into symbiont roles, physiology, and interactions within the holobiont. Under this premise, we aimed to isolate and culture bacteria from four Mediterranean corals. The recovery of 84 pure bacterial isolates and their initial classification based on the 16S rRNA gene revealed substantial diversity among symbionts amenable to culture. Several isolates represent novel species within relevant genera, such as Vibrio, underscoring the value of culture-based studies. All cultures were cryopreserved to guarantee long-term accessibility for future projects. This represents a key step towards describing the roles of bacteria within the coral holobiont, as cultures enable in-depth morphological and physiological characterization of the symbionts and experimental ecology studies.